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Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria.

Straub SV, Giovannucci DR, Yule DI - J. Gen. Physiol. (2000)

Bottom Line: Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import.This effect was also abolished by ryanodine receptor (RyR) blockade.Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.

ABSTRACT
In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

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Inhibition of RyRs diminishes global [Ca2+]i increases resulting from photolysis of caged InsP3. (A I and B I) Photolysis of 2 μM caged IP3 under control conditions evokes a global [Ca2+]i increase that initiates at the apical trigger zone and spreads as a wave throughout the cell, as shown by the [Ca2+]i traces in A and OGB-2 fluorescence images in B. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (A, II–IV, and B, II–IV) After addition of ryanodine to the bath solution, the propagation of the [Ca2+]i increase into the basal region decreases with each subsequent uncaging, such that the increase in [Ca2+]i is only evident within the granule-containing region of the cell. (A V and B V) Stimulation with a high dose of CCh is able to overcome the effects of blocking RyR, resulting in a global [Ca2+]i increase. (C) Pooled data showing distance [Ca2+]i wave propagated, plotted as a percentage of the first flash value, for control and ryanodine-treated cells. For control, n ≥ 9 for ryanodine treated, n = 7. *Statistically significant, P < 0.04. (D and E) Control uncaging of 2 μM caged InsP3. InsP3 was photolyzed every 3 min. Traces and fluorescence images correspond to time points I and IV in A, respectively, illustrating no significant run down of the response over this time period.
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Figure 8: Inhibition of RyRs diminishes global [Ca2+]i increases resulting from photolysis of caged InsP3. (A I and B I) Photolysis of 2 μM caged IP3 under control conditions evokes a global [Ca2+]i increase that initiates at the apical trigger zone and spreads as a wave throughout the cell, as shown by the [Ca2+]i traces in A and OGB-2 fluorescence images in B. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (A, II–IV, and B, II–IV) After addition of ryanodine to the bath solution, the propagation of the [Ca2+]i increase into the basal region decreases with each subsequent uncaging, such that the increase in [Ca2+]i is only evident within the granule-containing region of the cell. (A V and B V) Stimulation with a high dose of CCh is able to overcome the effects of blocking RyR, resulting in a global [Ca2+]i increase. (C) Pooled data showing distance [Ca2+]i wave propagated, plotted as a percentage of the first flash value, for control and ryanodine-treated cells. For control, n ≥ 9 for ryanodine treated, n = 7. *Statistically significant, P < 0.04. (D and E) Control uncaging of 2 μM caged InsP3. InsP3 was photolyzed every 3 min. Traces and fluorescence images correspond to time points I and IV in A, respectively, illustrating no significant run down of the response over this time period.

Mentions: To investigate whether CICR occurred during normal signaling events, and not solely when mitochondrial Ca2+ import was inhibited, a higher concentration of caged InsP3 (2 μM) was used to photolytically induce global [Ca2+]i increases. This allowed for the direct assessment of the role of RyR on the propagation of global Ca2+ waves. Using this paradigm, global [Ca2+]i increases could be reproducibly evoked (Fig. 8D and Fig. E). After release of InsP3 under control conditions, 100 μM ryanodine was added to the external bath solution. In the continued presence of ryanodine, sequentially evoked [Ca2+]i responses became progressively more confined to the apical region of the cell compared with control (Fig. 8A and Fig. B, n = 7). The addition of ryanodine not only prevented the [Ca2+]i increase throughout the basal region, but also caused the signal to retreat well into the apical region, giving increases in a region only slightly more diffuse than the trigger zone. By the fourth uncaging, the [Ca2+]i wave propagated 11.2 ± 1.8 μm (n = 7) in the presence of ryanodine, compared with 15.7 ± 1.3 μm in time-matched controls (Fig. 8 C, n = 10, P = 0.006). This decrease in propagation represented a reduction in the distance traveled across the cell from 95.5 ± 2.9% of the diameter of the cell to 63.9 + 10.9% in the presence of ryanodine. In addition, the rate of propagation was significantly slowed in the presence of ryanodine, traveling at 21.2 ± 6 μm/s (n = 10) in the absence of and 9.5 ± 2.3 μm/s (n = 7) after incubation in ryanodine for 12 min (P = 0.03). The latency after the flash to the initiation of the signal was not significantly altered by treatment with ryanodine (192 ± 5 vs. 175 ± 2 ms, control vs. treated after 12 min, n = 4), as expected for a signal initiated through InsP3R. Interestingly, the spatially limiting effects of RyR blockade could be overcome at higher agonist concentrations (Fig. 8A V and B V).


Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria.

Straub SV, Giovannucci DR, Yule DI - J. Gen. Physiol. (2000)

Inhibition of RyRs diminishes global [Ca2+]i increases resulting from photolysis of caged InsP3. (A I and B I) Photolysis of 2 μM caged IP3 under control conditions evokes a global [Ca2+]i increase that initiates at the apical trigger zone and spreads as a wave throughout the cell, as shown by the [Ca2+]i traces in A and OGB-2 fluorescence images in B. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (A, II–IV, and B, II–IV) After addition of ryanodine to the bath solution, the propagation of the [Ca2+]i increase into the basal region decreases with each subsequent uncaging, such that the increase in [Ca2+]i is only evident within the granule-containing region of the cell. (A V and B V) Stimulation with a high dose of CCh is able to overcome the effects of blocking RyR, resulting in a global [Ca2+]i increase. (C) Pooled data showing distance [Ca2+]i wave propagated, plotted as a percentage of the first flash value, for control and ryanodine-treated cells. For control, n ≥ 9 for ryanodine treated, n = 7. *Statistically significant, P < 0.04. (D and E) Control uncaging of 2 μM caged InsP3. InsP3 was photolyzed every 3 min. Traces and fluorescence images correspond to time points I and IV in A, respectively, illustrating no significant run down of the response over this time period.
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Related In: Results  -  Collection

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Figure 8: Inhibition of RyRs diminishes global [Ca2+]i increases resulting from photolysis of caged InsP3. (A I and B I) Photolysis of 2 μM caged IP3 under control conditions evokes a global [Ca2+]i increase that initiates at the apical trigger zone and spreads as a wave throughout the cell, as shown by the [Ca2+]i traces in A and OGB-2 fluorescence images in B. [Ca2+]i traces were generated from the apical and basal regions depicted in the brightfield image. (A, II–IV, and B, II–IV) After addition of ryanodine to the bath solution, the propagation of the [Ca2+]i increase into the basal region decreases with each subsequent uncaging, such that the increase in [Ca2+]i is only evident within the granule-containing region of the cell. (A V and B V) Stimulation with a high dose of CCh is able to overcome the effects of blocking RyR, resulting in a global [Ca2+]i increase. (C) Pooled data showing distance [Ca2+]i wave propagated, plotted as a percentage of the first flash value, for control and ryanodine-treated cells. For control, n ≥ 9 for ryanodine treated, n = 7. *Statistically significant, P < 0.04. (D and E) Control uncaging of 2 μM caged InsP3. InsP3 was photolyzed every 3 min. Traces and fluorescence images correspond to time points I and IV in A, respectively, illustrating no significant run down of the response over this time period.
Mentions: To investigate whether CICR occurred during normal signaling events, and not solely when mitochondrial Ca2+ import was inhibited, a higher concentration of caged InsP3 (2 μM) was used to photolytically induce global [Ca2+]i increases. This allowed for the direct assessment of the role of RyR on the propagation of global Ca2+ waves. Using this paradigm, global [Ca2+]i increases could be reproducibly evoked (Fig. 8D and Fig. E). After release of InsP3 under control conditions, 100 μM ryanodine was added to the external bath solution. In the continued presence of ryanodine, sequentially evoked [Ca2+]i responses became progressively more confined to the apical region of the cell compared with control (Fig. 8A and Fig. B, n = 7). The addition of ryanodine not only prevented the [Ca2+]i increase throughout the basal region, but also caused the signal to retreat well into the apical region, giving increases in a region only slightly more diffuse than the trigger zone. By the fourth uncaging, the [Ca2+]i wave propagated 11.2 ± 1.8 μm (n = 7) in the presence of ryanodine, compared with 15.7 ± 1.3 μm in time-matched controls (Fig. 8 C, n = 10, P = 0.006). This decrease in propagation represented a reduction in the distance traveled across the cell from 95.5 ± 2.9% of the diameter of the cell to 63.9 + 10.9% in the presence of ryanodine. In addition, the rate of propagation was significantly slowed in the presence of ryanodine, traveling at 21.2 ± 6 μm/s (n = 10) in the absence of and 9.5 ± 2.3 μm/s (n = 7) after incubation in ryanodine for 12 min (P = 0.03). The latency after the flash to the initiation of the signal was not significantly altered by treatment with ryanodine (192 ± 5 vs. 175 ± 2 ms, control vs. treated after 12 min, n = 4), as expected for a signal initiated through InsP3R. Interestingly, the spatially limiting effects of RyR blockade could be overcome at higher agonist concentrations (Fig. 8A V and B V).

Bottom Line: Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import.This effect was also abolished by ryanodine receptor (RyR) blockade.Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Physiology, University of Rochester, School of Medicine and Dentistry, Rochester, New York 14642, USA.

ABSTRACT
In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

Show MeSH
Related in: MedlinePlus